Extensive Association Analysis Between Polymorphisms of PON Gene Cluster With Coronary Heart Disease in Chinese Han Population

	Abstract

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Objective— An extensive association analysis of PON gene cluster (PONs) with coronary heart disease (CHD) was performed in Chinese Han population.

Methods and Results— Thirty polymorphisms of PON1, PON2, and PON3 gene were identified by direct sequencing of genomic DNA derived from 48 randomly selected patients. Twelve polymorphisms were additionally investigated for association with CHD in 474 male patients and 475 controls. Univariate analyses showed the cases had significantly higher frequencies of PON1 192Q allele, 160R allele, -162A allele, and PON2 311C allele than were seen in the controls. Logistic regression analyses revealed only the PON1 R160G and -162G/A polymorphisms remained significantly associated with CHD (P=0.0054 and P=0.0002). Haplotype analyses for various polymorphism combinations additionally confirmed the results of individual polymorphism analyses. Only the frequencies of haplotypes containing -162A allele were significantly higher, whereas only the frequencies of haplotypes containing 160G allele were significantly lower in cases than in controls in various polymorphism combinations.

Conclusions— This extensive association study has identified the PON1 -162G/A and R160G polymorphisms to be independently associated with CHD in Chinese Han population and warrants additional study to elucidate the biological mechanism.

Key Words: coronary heart disease  genetics  paraoxonase  linkage disequilibrium  LDL oxidation

	Introduction

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The oxidative modification of LDL in the artery wall is presently believed to be central to the pathogenesis of atherosclerosis. It was hypothesized that paraoxonase (PON1), an enzyme located on HDL, can prevent LDL from oxidation.¹ Substantial in vitro evidence supports such a function.^2,3 Two other members of the paraoxonase gene family, termed PON2 and PON3, have been identified. All 3 PON genes are located adjacent to each other on chromosome 7q21.3-q22.1.⁴ Recent studies show that human PON3 is also an HDL-associated enzyme with biological activity similar to PON1.⁵ Unlike PON1 and PON3, PON2 is not located on HDL and is more widely expressed. In 2001, Ng et al⁶ demonstrated that PON2 possesses antioxidant properties similar to PON1 and PON3 and exerts its antioxidant functions at the cellular level.

The PON1-coding region contains 2 common polymorphisms, a leucine (L) to methionine (M) substitution at codon 55 and a glutamine (Q) to arginine (R) substitution at codon 192. The Q192R polymorphism seems to be the major determinant of serum PON1 activity toward various organophosphates⁷ and has been reported to affect the enzyme’s in vivo ability to hydrolyze oxidized lipids.⁸ The L55M polymorphism does not affect the catalytic activity against different organophosphates, but the PON1 55M allele is correlated with decreased mRNA and protein levels.⁹ Five polymorphisms in the PON1 5' regulatory region, named -107C/T, -126G/C, -162G/A, -831G/A, and -908G/C, were recently identified by 3 different groups. Cell-culture experiments with a reporter gene and genotyping studies in white populations established that these polymorphisms had a strong impact on gene expression and serum concentrations.^10–12 The PON2 gene has 2 common polymorphisms giving rise to amino acid substitutions of glycine (G) for alanine (A) at codon 148 and cysteine (C) for serine (S) at codon 311, and associations between PON2 polymorphisms, variation in plasma lipoprotein levels, and birthweight have been reported.^13,14 However, there are no reports on polymorphisms in the PON3 gene.

Numerous studies have been conducted to investigate whether people with the PON1 192R alloenzyme are more prone to coronary heart disease (CHD) than those with the Q alloenzyme.^15–17 Reviewing these studies reveals discrepancies, even in studies conducted in the same ethnic population. Some studies have also shown associations between the PON1 L55M or PON2 S311C polymorphism and atherosclerosis, although others have not.^18,19 Of note, the studies above just assessed one or two of the given single nucleotide polymorphisms (SNPs), without consideration of potential interactions with other unidentified functional polymorphisms in this gene cluster, so this variability in results might be attributable to different linkage relationships with other functional genetic variants in different populations. The aim of the present study was to scan the whole PON gene cluster to identify all putative functional polymorphisms and additionally to investigate whether these polymorphisms were associated with CHD in Chinese Han population.

	Methods

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Subjects
Four hundred seventy-four male patients with CHD were recruited from hospitalized patients of Fu Wai Hospital and Cardiovascular Institute (Beijing, China) between October 1997 and September 1999. Men who survived an acute myocardial infarction or documented by coronary angiography at least a 70% stenosis in a major epicardial artery were eligible. Subjects with congenital heart disease, cardiomyopathy, valvular disease, and renal or hepatic disease were excluded from the study. Four hundred seventy-five age-matched male controls were randomly selected from subjects participating in a community-based survey of cardiovascular risk factors in Beijing. The control subjects were judged to be free of CHD by history, clinical examination, electrocardiography, and Rose questionnaire.

A set of questionnaires was completed that included details of medical history, family history of CHD, drug intake, cigarette smoking, and alcohol consumption. Blood pressure, weight, height, waistline, and hip circumference were recorded, and the body mass index and waist-to-hip ratio were calculated.

Venous blood was drawn from all subjects after an overnight fast. Blood, serum, and plasma were separated immediately and stored at -70°C.

Analysis of PON1 Activity
One hundred fifty cases and the same number of controls were randomly selected from the subjects for PON1 activity measurement. PON1 activity toward phenylacetate was monitored at 270 nm for 2 minutes¹² (Hewlett Packard 8453 spectrophotometer), and results are expressed as OD min^-1/mL^-1. Samples were arrayed twice, and the average was recorded. The intraassay and interassay coefficients of variation were 1.9% and 3.1%, respectively.

Screening of the Genes
Common polymorphisms of PON1, PON2, and PON3 gene were identified by direct sequencing of genomic DNA derived from 48 randomly selected patients. We designed PCR arrays to amplify regions up to 1 kb upstream from transcription-initiation sites, ie, putative promoter regions, all exons, and adjacent noncoding regions. Fluorescent dye-terminator cycle sequencing was performed, and products were analyzed with an Applied Biosystems 3700 capillary sequencer.

Genotyping
Twelve polymorphisms were genotyped in all 949 subjects. PON1 promoter region polymorphisms including -107, -126, -162, -831, -908, and -1076 were genotyped by sequencing PCR products. PON2 A148G and S311C polymorphisms were genotyped by allele-specific PCR amplification. PON1 L55M, R160G, and Q192R, as well as PON3 -133C/A polymorphisms, were genotyped by PCR-RFLP. The primers, lengths of PCR products, related restriction endonuclease, as well as digested bands are shown in the online Table (available at http://atvb.ahajournals.org.).

Statistical Analysis
The data were analyzed using the SAS statistical software, 2LD program (University of London, http://www.iop.kcl.ac.uk/IoP/ Departments/PsychMed/GEpiBSt/software.stm) and EH program (Rockefeller University, http://linkage.rockefeller.edu).

All data are presented as mean±SD. Hardy-Weinberg equilibrium was assessed by the ² test. Pairwise linkage disequilibrium coefficients were calculated from estimated haplotype frequencies using 2LD program. The extent of disequilibrium was expressed in terms of D'. Univariate analysis used to measure the association of each single polymorphism with CHD was tested by ² test. Multivariate analysis applied to investigate the independent role of each polymorphism was done by stepwise logistic regression. Odds ratios were computed from regression parameters. Haplotype frequencies for various polymorphism combinations were estimated by EH program. Two haplotype-based hypothesis tests were conducted. The first, an omnibus likelihood ratio test, which examines the differences in haplotype frequency profiles between the case and control groups, was analyzed by EH program. The second, examining the differences in individual haplotype frequencies between the cases and controls, was assessed by ² test based on the frequency of each haplotype versus all others combined.

No correction for multiple testing was applied, because it was not clear how many independent tests should be taken into account. Polymorphisms were not statistically independent because of strong linkage disequilibrium. The significance level for statistical tests was taken to be 0.05.

	Results

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Characteristics of the Subjects
Of the 474 cases, 4.5% were receiving a diuretic, 6.7% were taking a ß-adrenoceptor–blocking drug, 22.5% were receiving a calcium channel blocker, 9.7% were taking an ACE inhibitor, and 23.2% were taking lipid-lowering medication. All patients were receiving aspirin (50 mg daily).

The demographic details of the cases and controls are given in Compared with the control group, the CHD group had a greater proportion of smokers, more patients with hypertension, diabetes, or stroke, and larger average body mass index, waist-hip ratio, and systolic blood pressure. The CHD group also had significantly higher serum triglyceride levels, LDL cholesterol levels, and fasting glucose levels and lower HDL cholesterol than the control group. However, diastolic blood pressure was significantly lower in cases than in controls, which could be the result of medication in the cases after they were diagnosed. Among the randomly selected 150 cases and 150 controls, no significant difference was found in the serum PON1 activity 

fig.ommitted Comparison of Characteristics Between Cases and Controls

Description of the Polymorphisms
In a total length explored of 13.9 kb, we identified 30 polymorphisms, 7 in promoter regions, 4 in coding elements, 14 in introns, and 5 in 3' untranslated regions. Besides the 5 polymorphisms having been reported in the PON1 promoter region, a -1076 A/G polymorphism in PON1 gene and a -133C/A polymorphism in PON3 gene were detected. -133C/A was located in a potential binding site for transcription factor LF-A1T. Besides PON1 Q192R and PON2 A148G and S311C polymorphisms, one novel polymorphism giving rise to amino acid substitutions of arginine (R) for glycine (G) at codon 160 (an adenine transition to guanine at nucleotide 108 of exon5) was identified in PON1 gene; however, PON1 L55M polymorphism, which is common in a white population, was not detected in our 48 patients.

The 11 polymorphisms located in coding and promoter regions of PON gene cluster as well as the PON1 L55M polymorphism were additionally genotyped in all subjects. Their positions and mutation types as well as allele frequencies and pairwise linkage disequilibrium coefficients in control population are presented in . No significant deviation from Hardy-Weinberg equilibrium was observed for any polymorphism. However, even in the whole sample, we did not detect any subject with M allele of PON1 L55M polymorphism. The -107C/T and -908 G/C polymorphisms were almost completely concordant (ie, same allele frequencies and almost complete positive linkage disequilibrium). The -107T allele was always carried by the -908C allele. Almost complete concordance was also observed between -126G/C, -162G/A, and -1076A/G polymorphisms (2% recombinant haplotypes) and between A148G and S311C polymorphisms (1% recombinant haplotypes). Three groups of tightly associated polymorphisms were thus defined, hereafter referred to as {-107C/T, -908G/C}, {-126G/C, -162G/A, -1076A/G}, and {A148G, S311C}. To avoid redundancy, only -107C/T, -162G/A, and S311C polymorphisms are reported in the following text.

fig.ommitted  Allele Frequencies and Pairwise Linkage Disequilibrium Coefficients Between the PON Gene Cluster Polymorphisms in Control Subjects

Polymorphisms Associated With CHD
Univariate analyses indicated that the PON1 Q192R, R160G, -162G/A, and PON2 S311C polymorphisms were significantly associated with CHD. The cases had significantly higher frequencies of PON1 192Q allele, 160R allele, -162A allele, and PON2 311C allele than were seen in the controls (). In multivariate analysis with CHD as the dependent variable and the 4 polymorphisms as independent variables, the PON1 R160G and -162G/A polymorphisms remained associated with CHD (P=0.0054 and P=0.0002, respectively), whereas the PON1 Q192R and PON2 S311C polymorphisms were not significant (P=0.92 and P=0.12, respectively). The  presented the PON1 Q192R and PON2 S311C genotype distributions as a function of PON1 R160G or -162G/A genotypes and illustrated that there were more 192Q allele carriers or 311C allele carriers in 160RR or -162AA genotype. This suggests that the 192Q allele and 311C allele were in linkage disequilibrium with the 160R allele or -162A allele in the study population and may explain why the cases had significantly higher frequencies of 192Q allele and 311C allele in univariate analysis.

fig.ommitted  Genotype Frequencies of PON Gene Cluster Polymorphisms in the Study Subjects

fig.ommitted   PON1 Q192R genotype and PON2 S311C genotype distributions as a function of PON1 -162G/A genotypes as well as a function of PON1 R160G genotypes

There was no linkage disequilibrium between the PON1 R160G and -162G/A polymorphisms, whether in cases or in controls (). Furthermore, the logistic regression analysis with both polymorphisms and their interaction as independent variables found their interaction term was not significant. This shows the R160G and -162G/A polymorphisms were independently associated with CHD. In multivariate logistic regression analysis, PON1 R160G and -162G/A polymorphisms remained significantly associated with CHD independent of traditional environmental risk factors (). Compared with the PON1 160 RR genotype, the adjusted odds ratio for the development of CHD in the PON1 160 G allele carriers (GG and GR genotypes) was 0.61 (95% CI, 0.41 to 0.91; P<0.05). The adjusted odds ratio was 1.77 (95% CI, 1.22 to 2.58; P<0.01) for the PON1 -162A allele carriers relative to the PON1 -162GG genotype.

fig.ommitted  Logistic Regression Analysis of Determinants of Coronary Artery Disease in Subjects

Haplotype Analyses
 displayed the results of haplotype analyses for several kinds of polymorphism combinations. The first combination contained the two polymorphisms showing independent association with CHD: the PON1 -162G/A and R160G. The second combination added the PON1 Q192R and PON2 S311C polymorphisms into the first combination. The third combination included all of the 7 polymorphisms. Both the 2-polymorphism combination and the 4-polymorphism combination resulted in significant omnibus haplotype profile tests. However, what is of extreme interest is the result of individual haplotype frequency comparisons. The cases had significantly higher frequencies of haplotypes containing -162A allele but significantly lower frequencies of haplotypes containing 160G allele versus the controls, and these features were observed in all kinds of polymorphism combinations (). This result was consistent with the individual polymorphism analyses.

fig.ommitted  Haplotype Frequency Estimates and Significance Levels of Case-control Comparison for Various Polymorphism Combinations

	Discussion

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The allele frequencies for PONs polymorphisms differ among ethnic groups. For example, the frequency of PON1 192 Q allele was reported to be 0.73 in white populations,¹² whereas it is 0.40 in our population. The M55 allele, which has a frequency of 0.35 in white population,¹² was not detected in our 949 male subjects. Besides the allele frequencies, the linkage pattern between polymorphisms also differs among ethnic groups. Take -107C/T and Q192R polymorphisms as an example: Brophy et al¹² observed the -107C allele was in linkage disequilibrium with the 192R allele in an American white population, whereas Leviev et al²⁰ and James et al²¹ indicated there was no linkage disequilibrium between these two polymorphisms in Swiss white population. On the contrary, the -107C allele was in linkage disequilibrium with the 192Q allele in our population. The interracial variability in the allelic frequencies for PONs polymorphisms and the different linkage pattern between polymorphisms strongly suggest that ethnicity-specific polymorphisms should be identified before association studies.

In our data, two groups of tightly concordant polymorphisms, {-107C/T, -908G/C} and {-126G/C, -162G/A, -1076A/G}, were identified in the promoter region of PON1 gene, of which, the {-126G/C, -162G/A, -1076A/G} group was significantly associated with CHD independent of traditional risk factors, with -162GA/AA genotypes being associated with higher risk (odds ratio, 1.77). Within a possible NF-1 transcription factor–binding site and having a minor effect on serum PON1 activity in a white population,¹² the -162 polymorphism is more likely to be functional than the -126 and -1076 polymorphisms. Interestingly, a recent publication has identified an interleukin-6 (IL-6)-responsive element that shows homology to the sequence, including the 3' of the -162 polymorphism.²² IL-6, a proinflammatory cytokine, has been shown to decrease PON1 expression in human HepG2 cells and PON1 activity in the plasma of mice. However, that was a short-term regulation and did not have a long effect on plasma PON1 activity. It has been suggested that the short-term regulation of PON activity mediated by IL-6, together with the IL-6 induction of MCP-1 in endothelial cells, may exacerbate the inflammatory reaction in an established atherosclerotic lesion and might be more important than the decreased plasma PON1 activity.²³ This was in agreement with our results. There was no difference in the serum PON1 activity between the cases and controls, nor in the -107 polymorphism, which has been established to have a predominant effect on plasma PON1 activity. Additional functional studies are required to explore what impact the -162G/A polymorphism has on regulation of PON1 activity mediated by IL-6.

A novel nonsynonymous polymorphism, PON1 R160G, was identified in our 48 patients. The polymorphism was significantly associated with CHD, with 160RG/GG genotypes being associated with lower risk (odds ratio, 0.61). No linkage disequilibrium and interaction effect between the PON1 R160G and -162G/A polymorphisms were found with 2LD program or stepwise logistic regression analysis. The pathogenesis of PON1 R160G polymorphism related to CHD is unclear. Our results showed that this polymorphism had no effect on both serum PON1 activity and HDL cholesterol level (data not shown). It is difficult to speculate whether R160G polymorphism has an effect on its protection against LDL oxidation without understanding the active site of serum PON1 and its physiological substrate.

An association between a polymorphism and a phenotype might be attributable to linkage disequilibrium with another position, as illustrated by PON1 Q192R and PON2 {A148G, S311C} polymorphisms. The linkage disequilibrium of 192Q allele and 311C allele with -162A allele or 160R allele resulted in significantly higher frequencies of 192Q allele and 311C allele in cases than in controls in univariate analysis. Without genotyping and adjusting the effects of the -162G/A and R160G polymorphism in multivariate analysis, we might draw the conclusion that the PON1 192Q allele or PON2 311C allele carrier is more prone to CHD. Therefore, just assessing one or two given polymorphisms without considering other putative functional polymorphisms and complex linkage relationships might be one reason for the discrepancies in association studies.

Haplotype analyses, which can overcome the loss of information attributable to biallelic polymorphisms and avoid the inflated type I error attributable to multiple comparisons, were thought to be more powerful than individual SNP analyses.^24,25 However, some researchers believe individual SNPs are more important. When we know little about the human history and disease region, both analyses should be considered.²⁶ In our study, the findings of haplotypes analyses additionally confirmed the results of individual SNPs analyses. In various polymorphism combinations, only the frequencies of haplotypes with -162A allele were significantly higher, whereas only the frequencies of haplotypes with 160G allele were significantly lower in cases than those in controls, regardless of the alleles of other polymorphisms.

In conclusion, this extensive association study has identified the PON1 -162G/A and R160G polymorphisms to be independently associated with CHD in Chinese Han population, which support the hypothesis that PONs variations are involved in the development of CHD and warrants additional study to replicate our results as well as elucidate the biological mechanism.

	Acknowledgments

 
This work was funded by grants 2001AA227081 and Z19-01-03-01 of The National High Technology Research and Development Program 001CB510207 of the National Key Basic Research Project, 2002BA711A05 and 2002BA711A08 of the National Tenth Five-Year Plan Key Programs from Ministry of Science and Technology of The People’s Republic of China and H010210370113 and H020220030130 Biomedical Project from the Council of Science and Technology, Beijing.

Received November 8, 2002; accepted November 22, 2002.

	References

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